Addition of Laponite to gelatin methacryloyl bioinks improves the rheological properties and printability to create mechanically tailorable cell culture matrices

Extrusion-based bioprinting has gained widespread popularity in biofabrication due to its ability to assemble cells and biomaterials in precise patterns and form tissue-like constructs. To achieve this, bioinks must have rheological properties suitable for printing while maintaining cytocompatibility. However, many commonly used biomaterials do not meet the rheological requirements and therefore require modification for bioprinting applications. This study demonstrates the incorporation of Laponite-RD (LPN) into gelatin methacryloyl (GelMA) to produce highly customizable bioinks with desired rheological and mechanical properties for extrusion-based bioprinting. Bioink formulations were based on GelMA (5%–15% w/v) and LPN (0%–4% w/v), and a comprehensive rheological design was applied to evaluate key rheological properties necessary for extrusion-based bioprinting. The results showed that GelMA bioinks with LPN (1%–4% w/v) exhibited pronounced shear thinning and viscoelastic behavior, as well as improved thermal stability. Furthermore, a concentration window of 1%–2% (w/v) LPN to 5%–15% GelMA demonstrated enhanced rheological properties and printability required for extrusion-based bioprinting. Construct mechanical properties were highly tunable by varying polymer concentration and photocrosslinking parameters, with Young's moduli ranging from ∼0.2 to 75 kPa. Interestingly, at higher Laponite concentrations, GelMA cross-linking was inhibited, resulting in softer hydrogels. High viability of MCF-7 breast cancer cells was maintained in both free-swelling droplets and printed hydrogels, and metabolically active spheroids formed over 7 days of culture in all conditions. In summary, the addition of 1%–2% (w/v) LPN to gelatin-based bioinks significantly enhanced rheological properties and retained cell viability and proliferation, suggesting its suitability for extrusion-based bioprinting.


I. INTRODUCTION
Three-dimensional (3D) bioprinting combines cells, molecules, and biomaterials to precisely pattern and assemble tissue-like structures. 1,23D bioprinting has been utilized in the fields of tissue engineering, regenerative medicine, and disease modeling to recapitulate key characteristics of various native tissues. 3A popular method of bioprinting is extrusion-based, where a bioink is forced through a nozzle to create controlled 3D constructs in a layer-by-layer fashion. 1,3ver the last decade, many biomaterials [e.g., gelatin methacryloyl (GelMA), polyethylene glycol (PEG), alginate, or collagen] have been developed into bioink formulations for bioprinting.The major challenges with adapting these materials for bioprinting lie in balancing the mechanical, rheological, chemical, and biological properties such the bioinks are both printable and biocompatible (i.e., within the "biofabrication window"). 4][10] To mitigate this limitation, temperature-controlled print beds and syringe barrels can be used.][16][17][18][19][20][21] Given the central role of rheology in the success or failure of a bioink, thorough rheological analysis is an efficient pathway to determine the suitability of prospective bioinks for extrusion-based printing. 11Several key rheological parameters should be considered during ink development, including viscosity, shear thinning, thixotropy, and temperature range required for stable bioprinting.Viscosity plays an important role in cell sedimentation, aggregation, and shear stresses during and after extrusion. 3,16Shear thinning is the non-Newtonian decrease in material viscosity with increasing shear rate resulting from the reorganization of polymer chains during the extrusion through the printing nozzle. 14,15Shear thinning bioinks can be deposited under lower extrusion pressures and lower shear stresses which may otherwise impact cell viability. 6,16,17Thixotropy refers to time-dependent shear thinning and can be associated with the viscoelastic properties of a material. 3,11,12,18In extrusion-based bioprinting, inks deposited onto desired surfaces require rapid recovery (non-thixotropic) and enhanced viscoelasticity to retain structural integrity with increasing layers being deposited.Together, these parameters influence yield stress, defined as the pressure required to overcome and initiate flow to dispense material. 19Another key parameter to consider during ink development is the temperature window required and is dependent upon biomaterial choice and printing outside of the window may result in reduced rheological properties and poor shape fidelity.Implementing strategies that facilitate shear thinning and nonthixotropic recovery enables increased printability whilst maintaining cellular viability. 5,15,20,21o advance 3D cell culture biomaterials into bioinks, various approaches have been explored.These include incorporating secondary polymers to enhance the main polymer formulations, utilizing supramolecular hydrogels, and forming nanocomposites. 143][24][25][26] For example, alginate and kappa carrageen lack cell adhesion ligands, and have poor cross-linking kinetics. 27While composites of GelMA and kappa carrageenan have been utilized to improved shape fidelity, the increased viscosity, shear stress and mechanical strength limit its tissue specificity. 28,29These modifiers improve shear thinning behavior and enhance print fidelity compared to non-modified bioink formulations, while maintaining similar cell viability and function. 8,26,30,31Temperature sensitivity and fluctuations still pose a limitation with current rheological additives with a focus shifted toward the introduction of temperature inert particles for enhanced stability and incorporation with currently available biomaterials.
3][34] Laponite (LPN), a smectite nanomaterial commonly used within the cosmetic industry, has been utilized as a rheological additive for osteogenic and vascular bioinks. 28,30,32,35LPN are diskshaped particles with a diameter of approximately 25 nm and thickness of 1 nm, and heterogeneity of its charge with negative face charges and positive rim charges. 32,36,37Once dispersed in aqueous solution, LPN nanoparticles self-organize via face-edge aggregation to form reversible thixotropic gels. 25,32,35LPN has been shown to enhance mechanical, and physical properties and contribute to extracellular matrix (ECM) remodeling. 25,35Previous studies have demonstrated LPN's capacity for drug and growth factor delivery, providing a customizable platform that can be tailored to each specific application, such as prolonged release of recombinant human bone morphogenetic protein 2 (rhBMP2) or transforming growth factor-b3 (TGF-b3), or tetracycline delivery for periodontal disease. 38,39e hypothesized that adding LPN to GelMA would alter shearthinning, temperature sensitivity and viscoelastic characteristics required for extrusion-based printing.Here, we demonstrate the incorporation of 1%-4% (w/v) LPN to 5%-15% (w/v) GelMA to enhance its rheological properties and printability in extrusion-based bioprinting.The concentrations of 0%-4% (w/v) LPN were selected based on previous studies, whereas 5%-15% (w/v) GelMA was selected based on the versatile tissue stiffness (elastic modulus) that can be matched, such as endothelial ($1 kPa) to musculoskeletal tissues ($150 kPa) for the development of multi-tissue specific bioinks. 25,29,32,35,40,413][44][45] In contrast, the current study aims to narrow down this concentration range.It employs a comprehensive rheological approach to investigate the ideal LPN concentration for incorporation into gelatin-based bioinks, considering various rheological properties in detail.This study focuses on a comprehensive rheological analysis to investigate the addition of LPN to GelMA and its influence on shearthinning, temperature sensitivity, viscoelasticity, mechanical properties, and printability.Finally, MCF-7 human breast cancer cell viability and spheroid formation is assessed to determine GelMA-LPN's suitability for in vitro cancer models.

II. RESULTS
To determine LPN's influence on the rheological properties of GelMA bioinks, a series of rheological tests were undertaken.Bioinks demonstrated increased viscosity and shear-thinning behavior across all GelMA concentrations (5%-15% w/v) in the presence of LPN (1%-4%) compared to LPN-free controls [Figs.1(a-i)-1(a-iii)].A power law model was utilized to further evaluate LPN's influence using the n index and K (flow consistency index) [Eq.(1)].Measures of n index characterize flow behavior (Newtonian fluids ¼ 1, shearthinning < 1, and shear-thickening > 1) and K index refers to the viscosity gradient of a fluid to shear rate.The addition of 2% and 4% LPN across all tested GelMA concentrations (5, 10, or 15%, w/v), respectively, significantly increased shear-thinning capacity with an n index of <0.25 and increased K index ranging from 40 to 300 Pa s [Figs.1(a-iv) and 1(a-v)], suggesting LPN enhances GelMA's initial viscosity whereupon shear is applied resulting in shear thinning.
Thixotropic tests were performed to simulate the extrusion process and determine ink shape recoverability post-extrusion using low (1 s À1 ) and high (100 s À1 ) shear rate cycles.Thixotropic responses (time-dependent shear thinning) were observed in 5%-15% GelMA containing 1% LPN, while groups containing !2% (w/v) LPN exhibited non-thixotropic behavior [Figs.1(b-i)-1(b-iii)], suggesting the higher LPN concentration bioinks would better retain their shape post-printing.The absolute viscosity, which is referred to as the initial change of viscosity during recovery phase (1 s À1 ), was calculated to evaluate the material's viscoelastic and shear thinning properties.The GelMA controls (5%-10% w/v) elicited no significant change of viscosity during initial recovery phase, further demonstrating viscoelastic liquid characteristics and poor recoverability.15% (w/v) GelMA exhibited a similar thixotropic trend, however, viscosity increased because of the high polymer concentration resulting in sol-gel formation from prolonged shear To assess the cross-linking kinetics of GelMA-based bioinks with and without LPN, oscillatory rheological tests were performed with 405 nm photocrosslinking of bioinks initiated after 1 min.The storage FIG. 1. Viscosity profiles and 3-interval thixotropy test of GelMA-LPN bioinks.(a) Viscosity shear rate (0.1-1000 s À1 ) ramps of 5%-15% GelMA with the addition of 0%-4% (w/v) Laponite (LPN) (i)-(iii) were performed to assess viscosity and shear shear-thinning behavior using power law regression model n index (Newtonian ¼ 1, shear-thinning < 1, and shear thickening > 1), and K index (flow consistency index) (iv) and (v).(b) A 3-interval viscosity test during low shear (1 s À1 ) and high shear rate (100 s À1 ) cycles of 30 s intervals of 5%-15% GelMA with 0%-4% (w/v) Laponite (i)-(iii) investigated the viscoelasticity and shape recoverability, (iv) average recovery of viscosity refers to the change (%) of viscosity from initial preshear phase to post high shear recovery.(v) The absolute viscosity refers to the initial recovery viscosity measurement change once high shear rate has been applied.Mean values with error bands indicating standard deviation (SD), n ¼ 5 for all groups.Statistical differences were performed using two-way ANOVA with Tukey post-hoc test ( Ã P < 0.05; ÃÃ P < 0.01; ÃÃÃ P < 0.001).
and loss moduli increased across all groups upon light activation, greater change was observed in 5%-15% (w/v) GelMA controls further highlighting the transition from viscoelastic-liquid to solid, compared to 5%-15% GelMA with 1%-4% (w/v) LPN which already exhibited viscoelastic-solid behavior (storage modulus > loss modulus) prior to cross-linking [Figs.2(a-i)-2(a-iv)].The cross-linking kinetics, derived from the storage modulus and rate of propagation, 47 were investigated to determine the influence of LPN on the network formation.Bioinks containing 5%-10% GelMA with the addition of 1 or 2% LPN, respectively, elicited an increase in mechanical properties compared to 5%-10% (w/v) GelMA controls [Figs.2(b-i)-2(b-iii)].The final Young's moduli after 3.5 min of photocrosslinking of LPN-free controls were 1.61 6 0.16 and 27.7 6 1.17 kPa for 5% and 10% GelMA, respectively, and groups containing 2% LPN measuring 1.59 6 0.31 (5% GelMA) and 42.5 6 9.9 (10% GelMA) [Fig.2(b-iv)].The incorporation of 4% LPN in 10%-15% (w/v) GelMA, respectively, decreased mechanical properties and rate of storage modulus propagation (dG 0 /dt) compared to lower concentrations of 0%-2% The addition of 4% LPN increased cross-linking propagation within 5% GelMA and led to increased Young's modulus compared to 5% GelMA with 0%-2% LPN (w/v) [Figs.2(c-i The rheological investigation demonstrated that inclusion of LPN resulted in the enhancement of crucial rheological properties of GelMA that are necessary for extrusion-based bioprinting.These properties included shear-thinning behavior (n index 0.16), shape recovery (!82.5%), mechanical properties (0.26-73.10 kPa), and reduced temperature-dependent fluctuations of viscosity (tan d: 0.17-0.55).The study determined that 2% (w/v) LPN showed the most significant improvement in these properties.Following selection of 2% LPN as the best additive concentration for 5%-15% (w/v) GelMA, studies were commenced using different conical nozzles (22 G-400 lm to 27 G-200 lm) to assess their influence on print fidelity of simple lattice structures.The study revealed that the size of the nozzle had an impact on the print fidelity.Specifically, an overall decrease in fiber diameter and pore size was observed as the concentration of GelMA increased within nozzle sizes 25-27 G, in contrast to 22 G which resulted in the printing of inconsistent structures [Fig.3(a)].
An initial cytocompatibility study was carried out over 7 days to evaluate the cytocompatibility of 5% GelMA and 2% (w/v) LPN in 20 ll free-swelling droplets to determine LPN's impact on breast cancer cell line MCF-7's spheroid formation and metabolic activity (Fig. S2).The addition of LPN increased the formation of metabolically active spheroid and aggregates compared to 5% (w/v) GelMA control (Fig. S2).The addition of LPN affected fluorescent imaging of live and fixed samples, resulting in increased background fluorescence within GelMA-LPN groups and inconsistent acquisition of images using lasers 365, 561, and 633 nm (Fig. S3).As a result, FDA alone was utilized to visualized live cells.Next, a secondary study was conducted over 7 days to evaluate the cytocompatibility of printable ink concentration [10% GelMA with 2% (w/v) LPN] and the impact of nozzle size (22, 25, and 27 G) on MCF-7 cells with 1 min cross-linking time (E ¼ 11.07 kPa).Cell morphology changed over time, with individual cells forming rounded spheroids that increased in size and metabolic activity over time [Figs.4(a) and 4(b)].Despite variations in nozzle size, there were no significant differences in spheroid/and cell aggregate size whereas significant differences in metabolic activity was observed on day 7 [Fig.4

III. DISCUSSION
2][3] However, the progress of extrusion-based bioprinting is hindered by the requirement for specific bioink properties that permit the creation of tissue-like constructs with physiological complexity. 49To overcome these limitations, it is imperative to enhance the current bioink standards and expand the biofabrication window through the development of advanced bioinks that exhibit both high resolution and cellular compatibility. 1,3,5,14,15Rheological analysis provides a prescreening methodology for evaluating the potential of bioinks, determining if they possess the critical properties required for extrusion-based bioprinting.In the current study, we conducted a rheology-focused investigation to assess the influence of LPN on GelMA bioink formulations to reduce temperature sensitivity and improve printability within the biofabrication window.
Previous studies of GelMA-LPN (10% GelMA þ 2% LPN w/v) have shown enhanced mechanical properties with up to twofold increase in Young's modulus (35.3 6 1.5 kPa) after 1.5 min crosslinking compared to GelMA controls. 29The current study demonstrated comparable mechanical properties of (37.5 6 9.1 kPa) [Fig.2(b-ii)] for the same composition and cross-linking time; however, the addition of 4% (w/v) LPN concentration resulted in decreased Young's modulus (15.0 6 8.3 kPa) and cross-linking propagation [Figs.2(b) and 2(c)].This effect was even more pronounced at 15% GelMA, where the modulus of hydrogels decreased with LPN concentrations >1% (w/v) compared to lower concentrations of GelMA (5%-10% w/v).It is hypothesized that increases in LPN concentration partially inhibits cross-linking of 10%-15% (w/v) GelMA concentrations. 55It was evident in low (5% w/v) GelMA concentrations, the high LPN concentration increased Young's modulus and cross-linking propagation [Figs.2(b-i) and 2(c-i)].On the other hand, at high GelMA concentrations, the local concentration has already passed optimal, and the high LPN concentrations prevent the formation of a consistent crosslinked network [Figs.2(b-ii)-2(b-iv) and 2(c-ii)-2(civ)].Incorporating 2% LPN to GelMA 5%-15% (w/v) demonstrated Young's moduli ranging from $0.91 to 73 kPa [Figs.][42] Bioprinting GelMA without a rheological additive is possible; however, consistent shape fidelity is limited by printing conditions and GelMA concentration.All bioinks containing 2% LPN were found to be printable and maintained their shape at room temperature (25 C) except for 5% GelMA.It is expected that if the bioprinting temperature were raised to 37 C, the print fidelity would likely diminish due to the temperature-sensitive nature of GelMA.One strategy to improve shape fidelity to achieve sol-gel transition is to regulate printing temperatures, with temperatures ranging from 21 to 27.5 C, and a build plate temperature of 10 C have been shown to thermally gelate the deposited GelMA fibers following extrusion. 56,57However, the use of straight nozzles during printing has been associated with increased shear stress on cells compared to conical nozzles. 11,16,58In the past decade, bioprinting technology has advanced, and many commercially available printers are now capable of thermoregulating both syringe and build plates, as well as cross-linking during ink extrusion, between layers, or after printing.Addition of 2% (w/v) LPN to GelMA significantly reduced temperature-dependent fluctuations of bioink viscosity [Fig.2(c)], suggesting improved and more consistent printability. 3The electrostatic interaction of GelMA and LPN has been shown to influence thermal stability with increased zeta potential reported (À13 6 2.63 to À34.4 6 0.9 mV) with 0.5%-2% (w/v) LPN concentration compared to 5% GelMA control (À11.3 6 0.4 mV). 42The negative charge interaction promotes LPN particle dispersion within GelMA polymer solutions and reduced temperature fluctuations [Figs.2(d-i)-2(d-iv)].This is important as the bioink temperature may change during the printing process, potentially affecting print fidelity and reproducibility.Based on the rheological evaluation and preliminary printing tests, 10% GelMA with 2% (w/v) LPN was determined to possess the ideal rheological properties for extrusion-based bioprinting and offers a highly tunable range of mechanical stiffness via visible light cross-linking.
In this study, the nozzle size used for bioprinting (ranging from 22 to 27 G) had limited effect on the behavior of MCF-7 cells in the printed constructs.The metabolic activity increased in 25-27 G groups over 7 days compared to 22 G nozzle size, while total number of cells and spheroid formation remained consistent in all groups [Figs.4(a) and 4(b)].Increasing the photocrosslinking duration of 1 min resulted in an increased stiffness (11.67 kPa) and 14 mW/cm 2 light intensity limiting spheroid size and aggregate cell clusters (Fig. S1).The biocompatibility was further investigated and compared to 10% (w/v) GelMA droplets with matched stiffness values (1 kPa, Fig. S1).The study determined the bioprinting and addition of 2% (w/v) LPN enabled the generation of metabolically active spheroids and cell aggregates while 10% (w/v) GelMA control produced slightly larger spheroids (1315 6 216 lm) compared GelMA-LPN (1183 6 167 lm).
While GelMA-LPN demonstrates its suitability as a bioink, LPN does introduce some challenges.During fluorescence imaging of constructs, we observed a high level of unspecific background fluorescence throughout hydrogel construct stained with 4 0 ,6-diamidino-2-phenylindole (DAPI), PI or Alexa Fluor TM 633 phalloidin in 405 and 633 nm channels (Fig. S3).The selection of buffer to disperse LPN particles as a result of increased levels of ions including Na þ , Ca 2þ and Mg þ reduces particle dispersion previously characterized using x-ray diffraction (XRD), energy-dispersive x-ray (EDX) and thixotropic behavior. 44,50A variety of mixing methods and buffers were tested to reduce background fluorescence and improve homogeneity of LPN incorporation to GelMA (Fig. S4).Dissolution of lyophilized GelMA and LPN powder in 100 mM HEPES at 37 C using a magnetic stirrer overnight resulted in consistent fluorescence intensity in 488 nm channels throughout Teflon-cast and printed hydrogels using fluorescein isothiocyanate (FITC)-GelMA, suggesting good mixing (Fig. S4).The unspecific background fluorescence in GelMA-LPN constructs was persistent and more pronounced when the Laponite (LPN) particles were homogeneously dispersed throughout GelMA, rather than being clustered (Figs.S3 and S5).The formation of LPN within different aqueous fluids [de-ionized water, PBS, and fetal bovine serum (FBS)] influences its "house of cards" structure altering particle dispersion and hierarchical structure, as well as increased electrostatic interaction due to negatively charged surfaces and pH effects. 59,60Previous studies have investigated LPN's fluorescent and absorption interaction with organic dyes such as crystal violet and Nile Red and found LPN absorption-induced reactions and spectral shifts of intensity at 630 nm. 42,54,61,62Previous studies have established that LPN electrostatically interacts with GelMA's polymer chains, influenced by the positive amino acids and slightly negative carboxylic acid groups, which promotes enhanced particle distribution throughout polymer network. 52,63Similar background fluorescence was observed in 10% (v/v) PEGDA with the addition of 2% (w/v) LPN encapsulated with and without MCF-7 cells (Fig. S5).The cause of the background fluorescence remains to be determined.While this poses an issue for traditional live/dead staining, a combination of live-cell stain (FDA or Calcein AM) and brightfield images, or live cell tracking dyes have been previously shown effective alternatives and polymerase chain reaction, RNA sequencing type assays have been minimally affected. 25,35,37,60While GelMA-LPN has demonstrated high cytocompatibility in our study with MCF-7 cells, this is a resilient cancer cell line which does not imply that it will be cytocompatible with all cell types.Other studies have shown cytocompatibility with pre-osteoblasts (NIH MC3T3), fibroblasts (NIH-3T3), epithelial (MIA PaCa-2) and human bone marrow stromal cells (hBMSC's), but further cytocompatibility studies using sensitive patient-derived cells are needed to expand GelMA-LPN's versatility as a bioink for extrusion-based bioprinting. 30,35,64he incorporation of a rheological factorial approach offers insight into light-activated bioink properties utilizing four main testing profiles: viscosity shear rate profile, 3-interval viscosity thixotropy, rotational oscillatory tests with visible light cross-linking and temperature profile, as an effective prescreening and concentration optimizer.It was revealed that the addition of LPN enhanced GelMA's critical rheological properties required for extrusion based bioprinting with LPN 2% (w/v) being the optimal additive concentration.The inclusion of LPN to GelMA increased its printability and allowed for culture of metabolically active spheroids.This study illustrated an effective rheological approach for the characterization of GelMA-LPN bioinks which enhanced GelMA's biofabrication window while maintaining high cellular viability.The current study has also narrowed the LPN concentration range previously utilized with gelatin-based bioinks to 1%-2% (w/v) LPN for extrusion-based bioprinting, based on crosslinking inhibition and negative effects on mechanical properties at 4% (w/v) LPN.Future research should further explore GelMA-LPN's physiochemical interactions and LPN concentration range for tissuespecific applications.

B. Rheology
Rheological measurements were performed using a MCR302 rheometer (Anton Paar, Graz, Austria) through a series of rotational and oscillatory tests with cone-plate (CP) (25 mm diameter, 2 cone angle, 60 lm truncation height, and 0.05 mm fixed gap) and parallel-plate (PP) (25 mm diameter and 0.15 mm fixed gap).All testing was performed at 25 C unless otherwise stated.A CP geometry was utilized for viscosity profiles using a logarithmic shear rate ramp to determine shear-thinning properties.To determine the materials linear viscoelastic range (LVER) logarithmic ramps of strain and frequency (0.1%-1000% or 0.1 Hz) were performed [Fig.S1(a)].3-interval thixotropy test (3TT) viscosity and rotational oscillatory tests (photorheology and temperature profile) were performed within each sample's LVER range.Photorheology was performed using a LED light (405 nm) source placed at a set distance (6 cm for 32 mW/cm 2 and 14 cm for 14 mW/cm 2 ) from the rheometer plate [Figs.S1(b) and S1 (c)].A light intensity meter (Volumetric, BICO, Gothenburg, Sweden) was used to measure intensity prior to loading precursor solution.The shear-thinning properties were further evaluated using a power law equation ( 1) to define flow behavior, and Young's modulus (2) was calculated for photorheology experiments, where K (Pa s) is the consistency index and n is the power law index, where 2G Ã (Pa) is the complex shear modulus and v is the Poisson's ratio (GelMA ¼ 0.44). 7,46 Cell culture The MCF-7 human breast cancer cell line (CellBank, Sydney, Australia) was cultured in RPMI supplemented with 10% FBS, 1% (v/v) penicillin-streptomycin (Gibco, Thermofisher), 1% (v/v) MEM Non-Essential Amino Acids Solution (Gibco, Thermofisher), 1% (v/v) Sodium Pyruvate (Gibco, Thermofisher) and 0.1% (v/v) Insulin transferrin-selenium (Gibco, Thermofisher).MCF-7 cells were seeded in T75 flasks (Nunc, Thermofisher) and cultured to 70%-80% confluency in a cell culture incubator (37 C, 5% CO 2 ) and monitored daily using brightfield microscopy (Nikon) to assess morphology, with media changes performed every 2-3 days.

D. 3D cell culture
In all experiments, MCF-7 cells were used between passages 19-20.Cells were washed with PBS prior to detachment with 0.25% (v/v) trypsin EDTA (Gibco, Thermofisher) and counted using an automated cell counter (Invitrogen, Thermofisher) with 0.4% (v/v) trypan blue solution (Gibco, Thermofisher).MCF-7 cells were encapsulated in 5%-10% (w/v) GelMA with or without 2% (w/v) LPN to produce 20 ll free-swelling droplets with a cell density of 1 Â 10 6 cells/ml and cultured in 48-well plates (Thermofisher).All hydrogels were crosslinked for 30 s or 1 min to match stiffness using the LunaCrosslinker TM Visible Light Crosslinking System (Gelomics Pty Ltd), submerged in cell culture media and incubated at 37 C with 5% CO 2 .

E. 3D printing
A BIO X (CELLINK, Gothenburg, Sweden) 3D Bioprinter was used for all printing of bioink formulations at room temperature (RT) (25 C).GelMA-LPN formulations were prepared the day prior and heated to 37 C for 1 h before loading into a 3 ml syringe (Nordson, Westlake, Ohio, United States).All construct designs were developed using Inventor Professional (Autodesk) or custom G-codes.Files were then exported as .stlfiles and sliced using BioCAD (BIO X software, CELLINK).Printability index of selected bioinks was assessed using 15 Â 15 Â 3 mm 3 lattice structures with various infill densities (6-15%) and printed using smooth flow tapered nozzles 22 (400 lm), 25 (250 lm), and 27 G (200 lm) (Nordson).Once printed, each construct was crosslinked using the LunaCrosslinker (Gelomics Pty Ltd) for 1 min and then imaged using a stereomicroscope (Nikon, Tokyo, Japan); images were analyzed using Image J (National Institutes of Health, Bethesda, MA, USA) to quantify fiber diameter, pore size, and printability (Pr) where L is internal perimeter and A is internal area of each pore. 18or cell studies, a mixing adaptor 13 was 3D-printed using an Asiga PRO 4K digital lithography printer (Alexandria, Australia) with DentaGuide resin (Asiga) and sterilized with 70% w/v ethanol for 30 min and dried prior to achieving a homogenous mixture of bioink and cells.GelMA (10.8%) and (LPN 2.5%) were heated to 37 C and loaded into a 3 ml syringe (Terumo TM , Thermofisher).MCF-7 cells were prepared at the same cell density as 3D hydrogel models (3 Â 10 6 cells) in 200 ll of culture media and loaded into 3 ml syringe at 1:15 dilution to achieve final bioink concentration of 10% GelMA with 2% (w/v) LPN and final cell concentration of 1 Â 10 6 , using a 3D-printed mixing unit to achieve homogeneity and loaded into 3 ml cartridge.The bioinks were printed with tapered conical nozzles (22, 25, and 27 G) at room temperature (25 C), extrusion speeds of 12-20 mm/s, a pressure of 70-85 kPa and 15% infill density.Scaffolds of 10 Â 10 mm 2 size and 3-5 layers height were crosslinked post-printing using the LunaCrosslinker TM Visible Light Crosslinking System for 30 s.

G. Metabolic activity
The metabolic activity of MCF-7 in free-swelling and 3D-printed constructs was quantified using PrestoBlue Cell Viability Reagent (Invitrogen, Thermofisher) following the manufacturer's recommendation.The reagent solution was prepared at a 1:10 ratio (PrestoBlue: cell culture media), 500 ll of reagent solution was aliquoted per well and incubated for 45 min.Triplicates of 100 ll from each sample were aliquoted into a 96-well plate, and absorbance was read at 590 nm using a microplate reader (BMG LABTECH, Ortenberg, Germany) using two different gains (500 and 900).
H. Fluorescence staining MCF-7 containing samples were fixed with 4% paraformaldehyde (Sigma Aldrich) at day 1 and 7, respectively, for 45 min at room temperature followed by blocking and permeabilisation using 5% (v/v) goat serum (Gibco, Thermofisher) and 0.01% (v/v) Triton X-100 (Sigma Aldrich) in PBS for a minimum of 2 h.First, samples were stained with Alexa Fluor 488 or 633 phalloidin (1:400 dilution; Invitrogen, Thermofisher) overnight at 4 C on a shaker.Next, samples were washed three times in washing buffer (PBS with 1% v/v goat serum and 0.01% v/v Triton X-100) over 8 h at 4 C on a shaker.Samples were then incubated with 4 0 ,6-diamidino-2-phenylindole (DAPI, 1 lg/ml, Thermofisher) and CellMask TM Orange (5 lg/ml, Invitrogen, Thermofisher) overnight at 4 C on a shaker.The following day, samples were washed for a further 8 h with washing buffer and stored in PBS at 4 C until imaging.

I. Statistical and data analysis
Statistical analysis was performed using GraphPad Prism v9.2.Statistical significance was determined using one-way or two-way analysis of variance (ANOVA), as appropriate, and followed by post-hoc tests (Tukey or Bonferroni multiple comparison tests).A p-value of less than 0.05 was considered statistically significant with asterisks denoting statistical value ( Ã P < 0.05; ÃÃ P < 0.01; ÃÃÃ P < 0.001).Interaction terms were listed in appropriate figures unless stated otherwise.All experiments were performed with a sample size of n ! 3 and described within each figure caption.Data are presented using bar graphs with mean 6 standard deviation (SD).
(b)].A viability assessment was performed, comparing lattice constructs printed using a 27 G nozzle of 10% GelMA with 2% LPN and free-swelling droplets of 10% (w/v) GelMA with matching stiffness (E ¼ 1 kPa) [Figs.4(c) and 4(d)].The initial cell size and metabolic activity between the GelMA droplet control and the printed construct were similar.At day 7, both groups demonstrated cell aggregate/spheroid formation resulting in reduce total cell number and increased metabolic activity compared to day 1, suggesting proliferation of MCF-7 cells [Figs.4(c) and 4(d)].